【作者】 万超；

【导师】 张楚瑜； 潘兹书；

【作者基本信息】 武汉大学 ， 微生物学， 2009， 博士

【摘要】 猪瘟(classical swine fever, CSF)是由猪瘟病毒(classical swine fever virus, CSFV)引起的、猪的一种高度致死性传染病,在世界许多国家和地区广泛流行,在国际兽医局制定的《国际动物卫生法典》中,猪瘟被列入A类16种法定传染病之一。我国也作为Ⅰ类动物疫病加以重点控制。自50年代以来,我国自行研制的猪瘟兔化弱毒疫苗(“C”株)的广泛应用,在控制猪瘟的爆发流行中发挥了重要作用。然而,猪瘟兔化弱毒疫苗五十多年的长期使用,并未能有效控制猪瘟的发生和传播。近年来,我国猪瘟发生呈明显上升态势,并出现诸如散发流行、慢性猪瘟、持续感染和妊娠母猪带毒综合征等新特点。病毒逃避机体的免疫清除和在猪体内局部的持续存留；疫苗制品质量问题；运输和保存不当导致疫苗效力降低或失效；以及疫苗株毒力的回复突变等是其主要原因。因此,在欧美一些国家已明确禁止使用猪瘟弱毒活疫苗接种。我国是养猪大国,猪瘟的发生和流行,是制约农村养猪业发展的瓶颈。为满足对猪瘟预防、控制和最终根除的需要,研制新一代抗猪瘟疫苗制品以替代传统猪瘟兔化弱毒疫苗,势在必行。本研究在对猪瘟病毒囊膜糖蛋白E2和Erns保护性抗原区域结构分析的基础上,利用RT-PCR技术,以猪瘟病毒石门(Shimen)株基因组RNA为模板,扩增E2和Erns抗原编码区序列,与组织纤溶酶原激活剂(tPA)信号肽cDNA序列融合,克隆到真核表达载体pcDNA3.1(+)中,构建重组质粒ptPAs/△E2/△Erns和ptPAs/△E2,以及使用E2信号肽构建pE2s/△E2/△Erns和pE2s/E2。转染PK15细胞,检测目的基因的表达。用所构建的重组质粒免疫BALB/c雌性小鼠,免疫3次,每次间隔2周,设pcDNA3.1(+)及PBS阴性对照,第三次免疫2周后采血,分离血清。分别以原核表达并纯化的E2或Erns蛋白为包被抗原,间接ELISA方法检测血清抗体水平；以纯化的E2或Erns蛋白为刺激原,MTT法检测小鼠脾淋巴细胞增殖情况；以猪瘟病毒Shimen株或刀豆蛋白A(ConcanavalinA, ConA)为刺激原,Elispot法检测IFN-y分泌情况。结果表明,除pcDNA3.1(+)和PBS对照组外,各重组质粒免疫后小鼠血清抗体水平持续上升,与对照组相比差异显著(p<0.05)。融合tPA信号肽DNA疫苗免疫组的血清OD492nm值与E2信号肽DNA疫苗免疫组相比差异显著(P<0.05)。猪瘟病毒Shimen株或E2或Ems蛋白刺激后,嵌合E2和Erns基因DNA疫苗免疫组小鼠脾细胞分泌的IFN-y和T细胞增殖效果高于单价DNA疫苗组(p<0.05),含有tPA信号肽组高于E2自然信号肽组,融合E2和Erns的ptPAs/△E2/△Erns和pE2s/△E2/△Erns组要优于只含有E2的单基因组。以上结果表明,在诱导免疫应答方面嵌合DNA疫苗要优于单价DNA疫苗,tPA信号肽组优于E2信号肽组。在小鼠实验的基础上,选取诱导免疫效果较好的ptPAs/△E2/△Erns免疫CSFV血清抗体阴性猪,pE2s/E2和pcDNA3.1(+)同时免疫作为对照,免疫3次,每次间隔2周,第三次免疫2周后,以105TCID50剂量的CSFV Shimen强毒株经肌肉注射途径攻击。观察猪体临床症状,记录猪体体温。并分别在攻毒后第0、4、8、12天后采血分离血清,以及采集鼻拭子和频死猪组织样品。中和试验检测血清中和抗体水平,间接ELISA检测血清抗体IgG水平,RT-PCR检测猪瘟病毒在组织样品和血清样品中复制情况等。结果表明,嵌合DNA疫苗免疫后产生的血清中和抗体水平、白细胞数量明显高于非融合DNA疫苗,pcDNA3.1(+)对照组攻毒后实验猪全部死亡,pE2s/E2免疫组部分猪死亡,而ptPAs/△E2/△Erns免疫组全部猪存活,说明具有tPA信号肽的嵌合DNA疫苗ptPAs/△E2/△Erns具有针对猪瘟病毒完全的免疫保护效果,而E2信号肽单基因DNA疫苗pE2s/E2只具有针对猪瘟病毒部分的免疫保护。在此研究基础上,以p干扰素TIR结构域衔接蛋白(TRIF)作为分子佐剂和重组质粒ptPAs/△E2/△Erns或pE2s/E2同时免疫BALB/c雌性小鼠,免疫3次,每次间隔2周。第三次免疫2周后采血分离血清,分别以原核表达E2或Erns为包被抗原,间接ELISA方法检测血清抗体水平；以猪瘟病毒Shimen株或刀豆蛋白A(ConcanavalinA, ConA)为刺激原,Elispot法检测IFN-γ分泌情况。结果表明,重组质粒ptPAs/△E2/△Erns或pE2s/E2与TRIF同时免疫后小鼠血清抗体水平持续上升,但与非佐剂组相比差异不显著(P>0.05)。猪瘟病毒Shimen株刺激后,TRIF和DNA疫苗共免疫组小鼠脾细胞分泌的IFN-γ数量明显高于非佐剂DNA疫苗组(p<0.05)。以上结果表明,TRIF共免疫可显著增强DNA疫苗诱导细胞免疫水平。以重组质粒ptPAs/△E2/△Erns和TRIF、pE2s/E2和TRIF免疫CSFV血清抗体阴性猪,免疫3次,每次间隔2周,第三次免疫2周后,以105TCID50剂量的CSFVShimen强毒株经肌肉注射途径攻击。分别于攻毒后第0、4、8、12天采血分离血清,中和试验检测血清中和抗体水平,间接ELISA检测血清抗体IgG水平,RT-PCR检测组织样品中CSFV分布、计算白细胞数量,观察猪体临床症状并记录猪体体温变化情况等。结果表明,ptPAs/△E2/△Erns和TRIF、pE2s/E2和TRIF共免疫DNA疫苗猪强毒攻毒后,白细胞数量明显高于非佐剂DNA疫苗,血清中和抗体水平与非佐剂组无明显差异,但DNA疫苗共免疫TRIF佐剂组猪只全部存活,说明TRIF能提高DNA疫苗的免疫效果,并诱导具有针对CSFV强毒株攻击的完全免疫保护。综上所述,本文首次构建了由CSFV主要抗原E2和Erns编码基因融合组成的抗猪瘟嵌合DNA疫苗,并对其在机体内诱导免疫效力进行了研究。在此基础上,研究了细胞通路信号分子TRIF共免疫对DNA疫苗免疫保护效果的影响。初步证实了抗猪瘟嵌合DNA疫苗可以诱导猪体增强的抵抗CSFV强毒株致死攻击的免疫保护,TRIF可显著提高DNA疫苗的细胞免疫应答和抗病毒作用,这些工作为新型抗猪瘟DNA疫苗的研制奠定了坚实的基础。更多还原

【Abstract】 Classical swine fever (CSFV) is a contagious disease of swine and leads to severe economic losses worldwide. According to "Animal Health Code", CSF was an OIE (Office International des Epizooties) listed disease. CSF was identified as class one contagious animal diseases in our country. From 1950s, hog cholera lapinized virus (Chinese "C" strain), a live attenuated CSF vaccine, was developed by our country and used to control CSFV spread. Though a live attenuated CSF vaccine is effective to control outbreak of CSF, a lot of new features of CSF such as sporadic epidemics, chronic infection, sustainable contagion and pregnancy with CSFV were recently observed. So, it is necessary to develop a new marker vaccine.In this study, E2 and Ems encoded sequences were amplified by RT-PCR using genomic RNA of CSFV strain Shimen as template, and then fused with nature E2 or tissue plasminogen activator (tPA) signal sequences to construct recombinant DNA vaccines, including ptPAs/△E2, ptPAs/△E2/△Erns, pE2s/△E2/△Erns and pE2s/E2. PK15 cells were transfected with the four plasmids to detect the expression of the foreign genes, respectively. The female BALB/c mice immunized with the recombinant plasmids for 3 times with 2 weeks interval. Antibody titers were tested by indirect ELISA using the purified E2 or Ems protein by prokaryotic expression as the coating antigen. CSFV or ConcanavalinA (ConA) was used as stimulus to detect splenocyte proliferation by MTT assay or to detect IFN-y secreting by Elispot assay. The results showed that the serum antibody titers in the four immunization groups were significantly increased compared to pcDNA3.1(+) group (p<0.05). Two weeks after the last inoculation mice immunized with plasmid fused with tPA signal exhibited significantly higher OD492nm than those immunized with nature E2 signal (p<0.05). After CSFV or protein stimulation, splenocyte isolated from mice immunized with chimeric DNA vaccine induced more IFN-γsecreting cells and enhanced spleen cell proliferation than that immunized with the single gene vaccine. Mice immunized with DNA vaccines fused with tPA signals induced better immune responses compared to that immunized with DNA vaccines with E2 signal.Serum-negative pigs were immunized with ptPAs/△E2/△Erns, pE2s/E2 and pcDNA3.1(+) separately with 3 times at a 2 week interval, for ptPAs/△E2/AEms has induced a better immune responses in mice model and pE2s/E2 and pcDNA3.1(+) were used as control group. Two weeks after the last immunization, all the pigs were intramuscularly challenged with 3×105 TCID50 Shimen strain CSFV. The temperature and clinical symptom was monitored and recorded daily and the blood and nasal swab samples were collected on days 0,4,8,12 post-challenge. After challenge neutralizing antibody titers was evaluated by virus neutralization assay, IgG antibody titers were detected by ELISA. CSFV genomic RNA in tissue or serum samples was tested by RT-PCR. Our results demonstrated that the pigs vaccinated with chimeric DNA vaccine fused with tPA signal induced higher titer of neutralizing antibody than those vaccinated with native E2 vaccine. The pigs inoculated with pcDNA3.1 (+) were all dead, pigs immunized with pE2s/E2 were partly dead and pigs immunized with ptPAs/△E2/AErns were all survived.Adaptor protein TRIF as molecular adjuvant of DNA vaccines was investigated. Female BALB/c mice immunized with pRK-TRIF plasmid for 3 times at 2-week interval, and pRK was used as negative control. Antibody titers were detected by indirect ELISA using the purified E2 or Erns protein as the coating antigen. CSFV or ConA was used as stimulus to induce IFN-γsecreting. The results showed that the serum antibody titers were not significantly increased with TRIF. After CSFV or protein stimulation, splenocyte isolated from mice immunized with the mixture of DNA vaccine and TRIF induced more IFN-γsecreting cells and higher level of spleen cell proliferation than non-adjuvant DNA immunized mice.Serum-negative pigs were immunized with ptPAs△E2AErns and TRIF or pE2s/E2 and TRIF separately, with 3 times at a 2 week interval. Two weeks after the last immunization, all the pigs were intramuscularly challenged with 3×105 TCID50 Shimen strain CSFV. The temperature and clinlcal symptom was monitored and recorded daily. The blood and nasal swab samples were collected on days 0,4,8,12 post-challenge. After challenge neutralizing antibody titers were evaluated by virus neutralization assay, and IgG antibody titers were detected by ELISA and and CSFV genomic RNA in tissue or serum samples was tested by RT-PCR. The results showed that the pigs co-vaccinated with DNA vaccine and TRIF were more white cell numbers than non-TRIF vaccine. But pigs immunized with DNA vaccine and TRIF revealed similar neutralizing antibody titers compared to that immunized with DNA vaccine alone. All of pigs co-immunized with DNA vaccine and TRIF survived.In summary, we first constructed chimeric DNA vaccines encoded E2 and Ems, and examined their efficacies in mice and pigs. We also investigated the immune responses in mice and pigs co-immunized with DNA vaccine and TRIF. Our results demonstrated that chimeric DNA vaccine could induce higher humoral and cellular immune responses in mice and protection against CSFV in pigs. TRIF as molecular adjuvant could enhance the cellular immune responses of DNA vaccines. These results will be contributed to develop a novel DNA vaccine against CSFV.更多还原